ES2483801T3 - Crystalline form of pyrido compound [6,1-a] isoquinolin-4-one - Google Patents

Abstract

A crystalline polymorph (I) of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] - isoquinolin-3 (4H) -yl] ethyl} urea) having an X-ray powder diffraction pattern comprising characteristic peaks, in terms of 2θ, at 10.1º and 12.9º.

Description

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DESCRIPTION

Crystalline form of pyrido compound [6,1-a] isoquinolin-4-one

5 Background of the invention

The polymorphic behavior of drugs can be of crucial importance in pharmacy and pharmacology. Polymorphs are, by definition, crystals of the same molecule that have different physical properties as a result of the order of the molecules in the crystalline network. The differences in physical properties exhibited by the 10 polymorphs affect pharmaceutical parameters such as storage stability, compressibility and density (important in the formulation and manufacture of products) and dissolution rates (an important factor in determining bioavailability ). Differences in stability may be the result of changes in chemical activity (for example, differential oxidation, such as a dosage generates discolorations faster when it is formed by a polymorph than when it is formed by another polymorph) or changes mechanical (for example, the tablets are agglutinated during storage because the kinetically favored polymorph is thermodynamically converted to a more stable polymorph) or both (for example, the tablets of a polymorph are more susceptible to breakage at high humidity values) . As a result of differences in solubility / dissolution, in the extreme case, some polymorph transitions may result in loss of potency or, at the other extreme, toxicity. In addition, the

20 physical properties of the crystal may be important in processing: for example, a polymorph could be more susceptible to solvates or it could be difficult to filter and wash free impurities (i.e., particle shape and distribution could be different between one polymorph and another).

Each pharmaceutical compound has an optimal therapeutic blood concentration and a lethal concentration. The

The bio-availability of the compound determines the dosage intensity of the drug formulation necessary to obtain the ideal blood level. If the drug can crystallize in the form of two or more polymorphs that differ in terms of bioavailability, the optimal dose depends on the polymorph present in the formulation. Some drugs show a narrow margin between therapeutic and lethal concentrations. For example, chloramphenicol-3-palmitate (CAPP) is a broad-spectrum antibiotic that is known to crystallize in at least three polymorphic forms and one

30 amorphous form. The most stable form is commercialized, A. The difference in bio-activity between this polymorph and another form B is a factor of eight - which creates the possibility of fatal overdoses of the compound if administered unconsciously as form B due to alterations during the processing and / or storage. Therefore, regulatory agencies, such as the US Food and Drug Administration, have begun to establish strict controls on the polymorphic content of the active component in solid dosage forms. In general,

For drugs that exist in polymorphic forms, if a thermodynamically preferred polymorph different from the pure form has to be marketed, the regulatory agency requires batch-by-batch control. Thus, it is important, for both medical and commercial reasons, to produce and market the pure drug in its thermodynamically more stable polymorph, substantially free of other polymorphs favored from the kinetic point of view.

40 U.S. Patent Nos. 6,794,391, 7,378,424 and 7,105,663 describe a compound RPL-554 (N- {2 [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo -6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea).

image 1

It would be beneficial to provide a stable polymorph composition of RPL-554, which has the advantages over less stable polymorphs or amorphous forms, including stability, compressibility, density, dissolution rates, increased potency and absence of toxicity.

50 Summary of the invention

In one aspect, the invention provides a polymorphic form (I) of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo6,7-dihydro-2H-pyrimido [6 , 1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) (RPL-554). As a result of its unique stability properties, this polymorph provides a (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H

Pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) highly pure in its thermodynamically more stable polymorphic form.

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In another aspect, the invention provides a compound of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7dihydro-2H-pyrimido [6,1-a ] -isoquinolin-3 (4H) -yl] ethyl} urea), in the form of a solid crystal consisting of more than 99% by weight of N- {2 - [(2E) -2- (mesitylimino) -9 , 10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) yl] ethyl} urea),

5 at least 95% in polymorphic form of a thermodynamically stable polymorph (I) of N- {2 - [(2E) -2 (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H -pyrrhido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea), said polymorph (I) presenting the following structural parameters obtained by means of individual crystal analysis:

Wavelength 0.71073 angstrom Triclinic crystalline system Space group P-1 Unit cell dimensions a = 8.1246 (4) angstrom  = 91.583 (4) º. b = 11,4573 (5) angstrom  = 90,299 (4) º. c = 13.2398 (6) angstrom  = 99.628 (4) º. Volume 1214.56 (10) angstrom3 Z 2.

In another aspect, the invention provides a crystalline polymorph (I) of N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4oxo-6,7-dihydro-2H-pyrimido [6 , 1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) having a powder-shaped x-ray diffraction pattern comprising characteristic peaks, in terms of 2, at 10.1º and 12 , 9th.

In certain embodiments, said X-ray powder diffraction pattern further comprises characteristic peaks, in terms of 2, at 15.3 ° and 17.6 °.

In another embodiment, said x-ray diffraction pattern comprises at least 5 characteristic peaks, in terms of 2, selected from 6.4º, 10.1º, 12.6º, 12.9º, 13.6º, 14.2º , 14.7º, 15.3º, 15.4º, 15.8º, 17.0º, 17.6º, 18.9º, 20.9º, 22.4º, 22.8º and 28.7º.

In certain embodiments, the polymorph has a powder-shaped x-ray diffraction pattern substantially as shown in Figure 1.

In other embodiments, the polymorph has a differential scanning calorimetry trace that shows a maximum at about 248 ° C.

In several embodiments, the polymorph has a differential scanning calorimetry trace substantially as shown in Figure 4.

In another aspect, the invention provides a solid composition comprising the polymorph as described herein.

In one embodiment, the invention provides a composition where at least about 50% by weight of N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro -2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present as said polymorph.

In other embodiments, the invention provides a composition where at least about 70% by weight of N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro Total -2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present in said polymorph.

In other embodiments, the invention provides a composition where at least about 90% by weight of N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6.7- Total dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present in said polymorph.

In other embodiments, the invention provides a composition where at least about 97% by weight of N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6.7- Total dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present in said polymorph.

In another embodiment, the invention provides a solid composition comprising the polymorph as described above and, a pharmaceutically acceptable carrier.

In certain embodiments, the solid composition comprises the polymorph and one or more additional compounds.

In another embodiment, the additional compound is a known therapeutic substance.

55 In certain embodiments, the therapeutic substance is used to treat asthma, allergic asthma, pollen rhinitis, allergic rhinitis, bronchitis, chronic obstructive pulmonary disease (COPD), acute adult dyspnoea syndrome (SDAA),

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cystic fibrosis, skin disorders, atopic dermatitis, psoriasis, eye inflammation, cerebral ischemia or autoimmune diseases.

In other embodiments, the invention provides a solid composition where therapeutic substances 5 are used to treat asthma or COPD.

In certain aspects, the invention provides a process for preparing the polymorph of the invention comprising:

(to)

combine N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) il] ethyl} urea) with a solvent to form a mixture, and

(b)

heating at a temperature of about 50 ° C or above, for a time and under appropriate conditions for the formation of said polymorph.

In certain aspects, the invention provides a process for preparing the polymorph of the invention comprising:

(to)

combine N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) il] ethyl} urea) with a solvent to form a mixture,

(b)

 filtration of said mixture,

(C)

heating at a temperature of about 55 ° C or above, for a time and under appropriate conditions to form said polymorph; Y

(d)

 filtration and drying.

In certain embodiments, the solvent is DMSO, ethanol, methanol, isopropanol, hexanes, pentane, ethyl acetate, dichloromethane, or chloroform. In another embodiment, the solvent is DMSO or ethanol.

In another embodiment, said mixture is maintained at a temperature of about 50 ° C or above, for about 24 to 96 hours. In another embodiment, said mixture is maintained at a temperature of about 50 ° C or above, for about 72 hours. In other embodiments, said mixture is maintained at a temperature of about 55 ° C or above, for about 24 to 96 hours. In various embodiments, said mixture is maintained at a temperature of about 55 ° C or above it for about 72 hours.

In other embodiments, said mixture is dried under vacuum between 25 and 50 ° C. In another embodiment, said mixture is dried under vacuum at 40 ° C.

In another embodiment, the invention provides a polymorph as described above, prepared by means of the method comprising

(to)

combine N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) il] ethyl} urea) with a solvent to form a mixture, and

(b)

heating at a temperature of about 50 ° C or above, for a time, under appropriate conditions to form said polymorph.

In another embodiment, the invention provides a polymorph as described above, prepared by means of the method comprising

(to)

combine N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) il] ethyl} urea) with a solvent to form a mixture,

(b)

 filtration of said mixture,

(C)

heating at a temperature of about 55 ° C or above, for a time and under appropriate conditions to form said polymorph; Y

(d)

 filtration and drying.

In another aspect, the invention provides a crystalline polymorph for use in the treatment of asthma, allergic asthma, pollen rhinitis, allergic rhinitis, bronchitis, chronic obstructive pulmonary disease (COPD), acute adult dyspnoea syndrome (SDAA), cystic fibrosis , skin disorders, atopic dermatitis, psoriasis, eye inflammation, cerebral ischemia or autoimmune diseases, in a mammal that requires it to be a polymorph (I) of N- {2 - [(2E) -2- (mesitylimine) - 9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea).

In one embodiment, the mammal is a human.

The polymorphic form of RPL554 is less stable since it can be transformed, with varying ranges, into several polymorphic forms under certain conditions and can be transformed during storage. Large-scale production of the polymorphic form should be demanded more, in a manner more consistent with the

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cGMP specifications required in terms of purity as well as impurity profile. Therefore, the amorphous form presents development challenges of a uniform and reproducible micronized dry solid powder of RPL554 for certain training applications. At the same time Form I of crystalline polymorph can be more easily manufactured on a large scale GMPc to provide solid RPL554 as an active pharmaceutical ingredient (API) in a

5 more consistent and reproducible process. The purification and isolation processes for the crystalline polymorph Form I product are more appropriate for large-scale cGMP production. The above advantages result in the supply of product batches that, in a consistent manner, are within the required cGMP specifications. As shown in one aspect of the present invention, Form I of crystalline polymorph is the thermodynamically more stable polymorphic form of RPL554 and can also be expected to have the longest shelf life. This advantage should also be extended to the potential useful life of any commercial drug product of RPL554 containing Form I of stable crystalline polymorph in the form of API. In addition, Form I of crystalline polymorph is more appropriate for the development of a uniform and reproducible dry micronized solid RPL554 powder for certain pharmaceutical formulation applications.

15 The study of N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) il] ethyl} urea) (RPL554), presented in crystalline form (designated Form I), showed that the material was very flexible against changes and that Form I was within the limits of the study developed, the polymorphic form more stable of known material (see experimental).

An experimentation interval was applied in order to discover the existence of new pure polymorphs of the compound, which are: anti-solvent addition; work in saturated solution; suspension maturation; formal recrystallization; mixed solvent studies; and crystallization of aqueous system.

25 The generation of the amorphous phase was achieved and this is also used in some studies with similarly conclusive results, since Form I predominated with the exception of only a few experiments in which some change was introduced. In the cases in which some small-scale change was induced, attempts were made to scale the crystallizations in order to fully characterize the forms (pure and mixed), in order to determine whether this cage was due to solvation or An entity not solvated. In the majority of cases, due to the delicate nature of the process variables necessary to repeat said crystallizations and the total stability of the system, Form I was returned. The main exception was DMSO, where a high phase potential was observed. fusion in the form of a mixture with Form I, after the main fusion within the decomposition cycle for the material. It could also be seen that DMSO returned two other altered phases before the present escalation, each of them different, but finally representative of what, so

35 most likely, it was a solvate and / or less stable form.

Previous reports suggested that amorphous material could be generated by cooling by crushing a molten mass. Rapid evaporation from DCM provided a robust method, while cooling by crushing a melt simply resulted in decomposition (amorphous by means of XRPD, but <20% of product by means of HPLC analysis).

Because historically it has not been known whether the process from the material generated in its own way was amorphous or crystalline, solubility tests of these materials were repeated to act as a guide, demonstrating that the amorphous phase was more soluble.

45 Finally, it was found that Form I was thermodynamically more favored and apart, the manipulation of the crystalline or amorphous material could be achieved satisfactorily with reference to the present altered physics of insulated materials, for example, powder versus a crystalline appearance larger.

Of importance during the progression of this material, it was that the pressure applied resulted in the generation of what is presumed to be an amorphous material, as indicated by XRPD and DSC. The thermal profile of this material showed an exothermic crystallization and a subsequent fusion endotherm that was very close to that of Form I. This could be verified by repeating with more material and isolating the product after the exothermic event and checking by means of XRPD and HPLC

55

Brief description of the drawings

Figure 1: Comparison of the measured data of RPL554 (upper part) versus the simulated individual crystal data (lower part). Figure 2: Comparison of the expansion of the two theta intervals (5-13 º 2). Figure 3: RPL554 transmission XRPD profile. Figure 4: DSC / TGA trace for RPL554. Figure 5: XRPD comparisons of DMSO / heptane anti-solvent additions (CG1099). Figure 6: Comparison of XRPD of crystals of saturated solution from RPL554 and DMSO

65 (JN376A4). Figure 7: DSC overlap of RLP554 input and new form from saturated DMSO solution

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(JN376A4). Figure 8: Selected suspension maturation results showing Form I for all series. Figure 9: XRPD profiles for mixed aqueous crystallizations (experiment PF86).

5 Figure 10: Melt cooled by crushing Form I of RPL554 from 245 ° C (carried out as PF84). Figure 11: DSC profile for RPL554 cooled by crushing (from 245 ° C, PF84). Figure 12: DSC Profile for milled Form I of RPL554. Figure 13: Comparison of compressed sample XRPD versus RPL554 of original material (CG1099F). Figure 14: Comparison of DSC of amorphous material by means of pressure against DSC of RPL554. Figure 15: Comparison of DSC of RPL554 and a batch of amorphous RPL554 from DCM (JN376). Figure 16: XRPD analysis of RPL554 (GC1099 SM) and amorphous material (JN376F). Figure 17: Comparison of XRPD for PF89A1 (DMSO) with Form I of RPL554. Figure 18: Combined DSC / TGA for crystallization PF90 with DMSO scaling.

15 Figure 19: Comparison of XRPD of Form I of RPL554 with the mixed phase DMSO form (PF90). Figure 20: Conformation of results of RPL554 analyzed by means of x-ray diffraction. Figure 21: Hydrogen bonding involved in structure RPL554. Figure 22: Reason for packing for RPL554.

Detailed description of the invention

The study of N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 ( 4H) il] ethyl} urea) (RPL554), presented in crystalline form (designated as Form I), showed that the material was very flexible to changes and that Form I was, within the limits of the study carried out, the polymorphic form more

25 stable of known material. An experimentation interval was applied in order to test this, including said studies using amorphous material. Both phases, after production, showed that Form I predominated with the exception of only a few experiments in which a change was induced. In cases in which a small-scale change was induced, attempts were made to scale the crystallizations in order to fully characterize the forms (pure and mixed), to determine whether the change was due to solvation or a non-entity. solvated In most cases, due to the nature of the process variables necessary to repeat said crystallizations and the total stability of the systems, Form I was returned. The main exception was DMSO, where a high potential melting phase was observed. in the form of a mixture with Form I, after the main fusion within the material decomposition cycle.

Finally, Form I was shown as the thermodynamically favored phase and the manipulation of either the crystalline or amorphous material could be achieved with reference to the altered physical presentation of the isolated materials, for example, powder versus the larger crystalline appearance. In this base 3 test, crystallizations were started with the help of the appropriate growing material for the analysis of individual crystals and to complement the indexing and refining of the unit cells carried out with the data obtained from high quality powder.

X-ray diffraction is the ideal tool to obtain the fingerprint of the pharmaceutical product of interest. Ideally, dust data is related to a unique crystalline form with a unique XRPD. Due to the collapse of crystallographic information from 3D to 2D, a typical XRPD observes the overlapping of the peaks of

45 diffraction at two elevated levels of theta. Because of this, indexing a diffraction pattern (which refers the data to unit cell parameters) remains a challenging stage, although ample progress has been achieved in recent years.

The data presented herein refers potentially to correct unit cell parameters of RPL554. The software algorithm allows to obtain the unit cell settings with figures of merit. The higher these values, the greater the changes to obtain to obtain the correct unit cell.

X-ray powder diffraction (XRPD) PANalytical X´Pert PRO: diffraction patterns were collected

X-ray in powder form on a PANanalytical diffractometer using Cu K radiation (45 kV, 40 mA), - goniometer, focusing mirror, divergence slit (1/2 inch, 1.27 cm) , welding slots in both the incident beam and the divergent beam (4 mm) and PIXcel detector. The software used for data collection was X´Pert Data Collector, version 2.2f, and the data was presented using X´Pert Data Viewer, version 1.2 d.

Samples were processed under ambient conditions and analyzed by means of XRPO of transmission metallized paper, using the powder as received. Approximately 2-5 mg of the sample was mounted on the position 96 sample plate on a polyimide film support (Kapton, thickness of 12.7 µm). Data were collected within the range 3-40º 2 with a continuous scan (speed 0.146º 2 / s).

65 Differential Scanning Calorimetry (DSC): DSC data was collected on a PerkinElmer Pyris 4000 DSC. The instrument was verified for energy and temperature calibration using certified indium. One was placed

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Pre-defined amount in milligrams mg of the sample in a drilled aluminum bowl and normally heated at 20 ° C min-1 from 30 ° C to 350 ° C, or varied as dictated by experimentation. The instrumental control and analysis data were Pyris Software v9.0.1.0174.

5 Thermo-Gravimetric Analysis (TGA): the data was collected in a Pyris 1 TGA equipped with an automatic 20-position sampling device. The instrument was calibrated using certified indium. A predefined amount in milligrams of the sample was introduced on a pre-weighed aluminum crucible and heated at 40 ° C min-1 from room temperature to 400 ° C. A nitrogen purge was maintained at 20 ml min-1 on the sample. The instrumental control and data analysis was Pyris Software v9.0.1.0174.

Gravimetric Steam Sorption: Sorption isotherms were obtained using a Hiden Isochema moisture sorption analyzer (IGAsorp model) controlled by IGASorp Systems Software V6.50.48. The sample was kept at constant temperature (25 ° C) by means of the instrumental controls. The humidity was controlled by means of dry and wet nitrogen mixing streams, with a total flow of 250 ml min-1. It was verified on

15 relative humidity content of the instrument measuring three calibrated Rotronic salt solutions (10-50-88%). The change in sample weight was monitored as a function of humidity by means of a micro-equilibrium (accuracy +/- 0.005 mg). A defined amount of sample was placed in a stainless steel container with previously weighed metal mesh under ambient conditions. A complete experimental cycle consisted of two sweeps (sorption and desorption) at a constant temperature (25 ° C) and intervals of 10% relative humidity over a range of 10-90% (90 minutes for each humidity level). This type of experiment should demonstrate the ability of the samples studied to absorb moisture (or not) with respect to a set of well-defined humidity ranges.

Nuclear Magnetic Resonance (NMR): 1H NMR spectra were collected on a Bruker 270MHz instrument

25 equipped with an automatic sampling device and controlled by a DRX400 console. Automated experiments were acquired using Delta RMN Processing & Control Software version 4.3. Samples were prepared in DMSO-d6, unless stated otherwise. The analysis was carried out using ACD / Specmanager 7.11.

Aqueous Thermodynamic Solubility by means of HPLC: the aqueous solubility was determined by suspending sufficient compound in HPLC quality water to provide a maximum final concentration of  20 mg ml-1 of the parental compound. The suspension was equilibrated at 25 ° C for 24 hours. Subsequently, the suspension was filtered through a filter into an HPLC vial. Subsequently, the filtrate was diluted by an appropriate factor. Quantification was performed by means of HPLC with reference to a standard solution of approximately 0.5 mg ml-1 in Acetonitrile: Water (1: 1). Different volumes of the standard sample solutions, diluted and undiluted, were injected. The solubility was calculated using the peak areas determined by means of integration of the peak found at the same retention time value as the main peak in the pattern injection.

The analysis was carried out on an Agilent 1100 series liquid chromatograph, equipped with a UV detector (DAD or VWD) @ 254 nm using the Chemstation Rev. B.01.03 software for data processing. Determination of chemical purity by means of HPLC.

Pharmaceutical Compositions

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a polymorph of the present invention formulated together with one or more pharmaceutically acceptable excipients. As used herein, the term "pharmaceutically acceptable excipient" means a liquid, semi-solid or solid, inert and non-toxic encapsulation, diluent or filler material or auxiliary formulation of any kind. The pharmaceutical compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (in the form of powders, ointments or drops), buccal or as oral or nasal spray. Other drug administration routes include inhalation of the medicament from a solution or suspension of the pharmaceutical compositions of the present invention in a controlled dosage inhaler (MDI) or neubulizer, or inhalation of a drug in powder form comprising a therapeutically amount effective of a polymorph of the present invention generally

Mixed with an excipient or pharmaceutical compositions of the present invention, from a dry powder inhaler (DPI).

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water, alcohol or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, polysorbate, dimethylformamide, oils (in particular, cottonseed oils, peanut, corn, germ, olive, castor and sesame), mono- or diglycerides, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, antioxidants, sweeteners,

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flavorings and scents. The liquid dosage form can also be encapsulated in a gelatin capsule, where a compound of the present invention can be dissolved in a pharmaceutically acceptable excipient containing, for example, one or more solubilizing agents (e.g., polysorbate 80 and mono and diglycerides) and other appropriate excipients (for example, antioxidants such as palmitate

5 ascorbyl, or a sweetening or flavoring agent).

Injectable preparations, for example, sterile injectable oleaginous or aqueous suspensions, can be formulated in accordance with known art, using appropriate wetting or dispersing agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable solvent or diluent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution,

U.S.P. and isotonic sodium chloride solution. In addition, sterile and fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any soft fixed oil including synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid are used in the

15 preparation of injectable formulations.

In order to prolong the effect of the drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved through the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, which, in turn, may depend on the size of the crystals and the shape of the crystals. Alternatively, delayed absorption of a drug form administered parenterally is achieved by dissolving or suspending the drug in an oily vehicle. Also contemplated are the immediate release forms by the present invention.

Preferably, the compositions for rectal or vaginal administration are suppositories that can be prepared by mixing the compounds of the present invention with appropriate non-irritating excipients or vehicles such as coconut butter, polyethylene glycol or suppository wax, which are solid to room temperature but liquids at body temperature and therefore melt in the cavity of the vagina or rectum and release the active compound polymorph.

Solid compositions of the similar type can also be employed as fillers in soft or hard filled gelatin capsules using such excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

35 The active polymorphs can also be in micro-encapsulated form with one or more excipients as discussed above.

Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and coatings such as enteric coatings, coatings that control release and other coatings well known in the art of pharmaceutical formulation. In said solid dosage forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as normal practice, additional substances other than inert diluents, for example, tablet-forming lubricants and other tablet-forming aids such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and

45 pills, the dosage forms may comprise buffering agents.

Preferably, a polymorph of the invention is formulated in a solid dispersion, where the polymorph can be molecularly suspended in a matrix comprising a pharmaceutically acceptable hydrophilic polymer. The matrix may also contain a pharmaceutically acceptable surfactant. Solid dispersion technology suitable for the formulation of a polymorph of the invention includes, but is not limited to, melt extrusion, spray drying or solvent evaporation.

Dosage forms for topical or transdermal administration of a polymorph of the present invention

55 include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers or patches. The active component is mixed under sterile conditions with a pharmaceutically acceptable excipient and any necessary preservatives or buffers that may be required. Also contemplated are the ophthalmic formulation, ear drops, eye ointments, powders and solutions within the scope of the present invention.

Ointments, creams, pastes and gels may contain, in addition to a polymorph of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid , talc and zinc oxide or mixtures thereof.

The powders and sprays may contain, in addition to the polymorphs of the present invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures thereof.

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substances Additionally, the sprays may contain usual propellants such as chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing a controlled supply of a polymorph to the

5 body Said dosage forms can be prepared by dissolving or supplying the polymorph in the appropriate medium. Absorption enhancers can also be used to increase the flow of the polymorph through the skin. The speed can be controlled either by providing a speed control membrane or by supplying the compound in a polymer matrix or gel.

Biological activity

In another aspect, the present invention provides a polymorph composition comprising a polymorph of the invention and a veterinarian or pharmaceutical acceptable diluent or excipient. Preferably, the composition is a pharmaceutical composition for human medicine. In another aspect, the composition of the

The present invention is a composition for veterinary medicine.

The polymorph compounds of the present invention are PDE inhibitors and thus possess valuable pharmacological properties, such as bronchodilator activity, as demonstrated by inhibition of field-stimulated contraction of trachea isolated in guinea pigs, and anti-inflammatory activity as illustrated in studies with human mononuclear cells stimulated by PHA (phytohemagglutinin). In vivo and in vitro data indicates that the compounds have a long-lasting action, as demonstrated by the persistent protective effects against histamine-induced bronchospasm in guinea pigs when direct inhalation in the lungs occurs in the form of dry powder. Therefore, the invention also relates to the acute, chronic and prophylactic treatment of patients suffering from respiratory disorders including, in particular,

25 asthma, allergic asthma, pollen rhinitis, allergic rhinitis, bronchitis, chronic obstructive pulmonary disease (COPD), acute adult dyspnoea syndrome (SDAA) and cystic fibrosis. Skin disorders such as atopic dermatitis and psoriasis, or ocular inflammation or any other disease including cerebral ischemia or autoimmune diseases, in which the increase in intracellular cAMP concentrations is considered beneficial, can also be treated topically.

One or more compound polymorphs may be present in association with one or more pharmaceutically and veterinarily acceptable non-toxic excipients and / or diluents and / or adjuvants and / or propellants and, if desired, other active ingredients. Appropriate excipients or diluents are known in the art (e.g. Handbook of Pharmaceutical Excipients (1994) 2nd edition, Eds. A. Wade / PJ Weller, The Pharmaceutical Press, American

35 Pharmaceutical Association).

According to another aspect, the present invention provides a composite polymorph for use in medicine. The polymorph compounds of the present invention are useful as inhibitors of phosphodiesterase isoenzymes. Polymorphs of the compounds or compositions of the present invention can be used to prevent or treat any disease in which the compounds or compositions are useful, but in particular a disease in which the increase in intracellular cAMP concentration is desirable. Examples of diseases against which the compounds are useful include respiratory disorders that include, in particular, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), acute adult dyspnoea syndrome (SDAA), allergic asthma, pollinic rhinitis, allergic rhinitis and cystic fibrosis. They can also be used topically in

45 skin disorders such as atopic dermatitis and psoriasis, eye inflammation or any other disease including cerebral ischemia or autoimmune diseases, in which the increase in intracellular concentrations of cAMP is considered beneficial.

This aspect of the invention is particularly relevant for the treatment of humans, but is applicable to the general veterinary industry, in particular domestic animals such as dogs and cats and farm animals such as horses, pigs, cows, sheep, etc.

Dosage levels of the order of about 0.02 mg to about 200 mg, taken three times a day, are useful for the treatment of the aforementioned disorders. More particularly, an interval

Dosing of about 0.2 mg to about 20 mg, taken three times a day, is effective. However, the particular dosage regimen will ultimately be determined by the attending physician and will take into account factors such as the medication used, age, weight, severity of symptoms and / or severity of the treatment being applied, methods of medication administration, adverse reactions and / or other contraindications.

The medication according to the present aspect of the invention can be provided to a patient together with other active agents, which may for example be a different compound or a polymorph of the present invention, or other compounds. Examples include adrenoceptor-2 agonists, topical glucocorticoid steroids, xanthine derivatives, antihistamine compounds, leukotriene antagonists, inhibitors of the synthesis of

65 leukotriene and / or combinations thereof.

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According to another aspect, the present invention provides the use of a polymorph of the invention in the manufacture of a phosphodiesterase isoenzyme inhibitor type III / IV (PDE3 / 4). The invention encompasses the use of a polymorph of the invention in the manufacture of a bronchodilator and / or an anti-asthmatic medication and / or a medicament for the prevention or treatment of chronic obstructive pulmonary disease (COPD).

The invention also relates to a crystalline polymorph for use in the treatment or prevention of a disease in a mammal, where a phosphodiesterase and / or bronchodilator isoenzyme inhibitor would be expected to be beneficial, the method comprising administering to said mammal an effective and non-toxic amount of a polymorph of the invention. The invention encompasses a crystalline polymorph for use in the treatment or

10 prevention of asthma and / or chronic obstructive pulmonary disease (COPD) in mammals.

Examples

The compounds and processes of the present invention will be better understood together with the following examples, which

15 are intended to be illustrative only and not limiting the scope of the invention. Various changes and modifications with respect to the described embodiments will be apparent to those skilled in the art and such changes and modifications, without limitation, those that refer to chemical structures, substituents, derivatives, formulations and / or methods of the invention, which are they can be carried out without departing from the scope of the appended claims.

twenty

Example 1

Approximately 50 mg of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2Hpyrimido [6,1-a] -isoquinolin-) 3 (4H) -yl] ethyl} urea) (RPL554) in 0.5 ml of DMF at 70 ° C. The solution was allowed to cool

Transparent yellow very slowly with nitrogen purge up to approximately 30 ° C for 4 days. An initial batch of material was observed within the liquors (approximately 0.4 ml) which was crystalline in nature. This process was repeated twice and appropriate material was supplied for the analysis of the individual crystals within a volume of liquor of approximately 0.2 ml. The sample was subjected to unaltered analysis within the maternal liquor.

30 The data of individual crystals were measured at low temperature (123 K) and at different wavelength (0.71073 angstrom). In order to provide a drawing that provides a direct comparison, manual adjustments of the wavelength value were necessary. After several adjustments, Figure 1 was obtained and shows the separation overlap between the measured data for RPL554 070638 by means of XRPD (upper part) and XRPD

35 simulated from the individual crystal data (Copy of onix2010a, bottom).

A closer inspection (Figure 2) in the range of two theta (5-13 ° C, 2) clearly demonstrates the similarities between the observed data and the simulated dust pattern.

40 The algorithm in the software compares numerically the observed data (RPL554 070638) measured by means of transmission XRPD and the numerical values from the individual crystal analysis. The key data from the individual crystal analysis are shown below:

Temperature 123 (2) K Wavelength 0.71073 angstrom Triclinic crystal system Space group P-1 Unit cell dimensions a = 8.1246 (4) angstrom  = 91.583 (4) º

b = 11,4573 (5) angstrom  = 90,299 (4) º

c = 13.2398 (6) angstrom  = 99.628 (4) º

Volume 1214.56 (10) angstrom3

Z 2

45 The software applied for comparison reads the measured file (RPL554 070638) and favored the input of the unit cell parameters extracted from the individual crystal data. Subsequently, Pawley's refining (algorithm) returns the numerical values, but the software also illustrates this numerical value by means of a graphical display of the result. The numerical value for the goodness of the fit between the measured and theoretical values for RLP554 was 2.9. This value expresses the quality of the data. The lower the value, the better the

50 data and, normally, a value less than 10 is considered a positive success.

The numerical values of RPL554 were in good tune with those from the individual crystal measured at 123 K. Thus, the individual crystal data provides an excellent fit with the dust data collected for RPL554 on its own and, therefore, is representative of the polymorph crystallographic profile

55 known thermodynamically favored (Form I).

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Example 2. Starting material

A sample of 9.0 g of RPL554 reserve ex-GMP (070638-9) is taken and analyzed by means of XRPD (Figure 3 and Table 1) and DSC (Figure 4) for use as a data set of current reference 5 Table 1 Pos. [º2 Th]

Height [beads]

6.3827 2406.64

10,1448 3293.52

12.6314 2495.28

12.8735 3362.54

13,6461 1682.38

14,2043 1714.62

14,7078 1191.12

15,2706 2566.43

15.4230 1230.41

15,8229 1877.13

16,9786 1303.49

17.6393 3211.22

18.8932 938.79

20,9592 1298.36

22.4382 1587.30

22,8201 1112.59

28.6947 876.91

Other studies such as proton NMR and HPLC at this juncture were not critical for the start of the project, since the results had already been collected previously. The studies of material solubility studies

10 using a range of common organic materials, likewise, is unnecessary since previous studies had provided an indication on the behavior of the material. The initiation of the investigation in the presence of other forms of RPL554 was considered a priority.

As is known, the material is crystalline, with a melting start of 246 ° C for the individual entotherm, without

15 later crystallization appreciated or transition, different from thermal decomposition. An initial weight loss of approximately 4.2% is appreciated by means of TGA, which corresponds to the fusion endotherm, which is not related to the solvent (probably loss of ammonia in front of total fragmentation). Having obtained the data, the experimentation with the material began, with the aim of subsequently producing the amorphous material. From this juncture, the above data is called Form I.

It is assumed that the process to produce RPL554 supplies the material in the current form and that the suspensions from alcohol used to improve its purity improve its crystallinity, or more importantly, convert the amorphous material into a crystalline form.

25 Example 3. Anti-solvent addition

The anti-solvent addition is a well known method to obtain the amorphous material, new polymorphs and also mixed phases. With this in mind, a stock solution of RPL554 in DMSO occurred. DMSO was chosen as the material that has a reasonable solubility in this solvent, which is also widely miscible.

30 Experimental conditions (CG1099):

300 mg of RPL554 was dissolved in hot DMSO (2 ml of stock solution) and 0.15 ml of this solution was added with intense stirring, in parts, on 12 cold tubes containing various solvents (1.0 ml) at temperature ambient. In almost all cases, instantaneous precipitation was observed. Isolation was carried out

35 by filtration and drying under vacuum at 40 ° C. The following Table 2 shows the results:

Table 2. List of solvents and details for anti-solvent additions based on DMSO

Solvent

Initial result Solids isolated

Cumene

soln  ppt F1

nBuOAc

ppt F1

Dioxane

soln F1 after drying

Water

ppt F1

Heptane

Bi-layer / oil  ppt F1

IPA

ppt F1

CH3CN

ppt F1

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Solvent

Initial result Solids isolated

MEK

ppt F1

ETOH

ppt F1

EtOAc

ppt F1

TBME

ppt F1

DCM

soln F1 after drying

All samples that returned the solid state, once dried under vacuum at 45 ° C and analyzed by means of XRPD showed the return of Form I. The notable exceptions were dioxane, DCM and heptane. Dioxane and DCM gave rise to solutions that were allowed to stand and evaporate, finally giving rise to Form I.

5 Heptane was more interesting in those two layers formed, both colored solutions, which gradually underwent oil formation after prolonged agitation and rest periods (DMSO layer). After approximately 24 hours, the solids formed in the opaque oil and, thus, the mixture was decanted and dried under vacuum at 45 ° C. Unfortunately, although the formation of the amorphous material could be expected, the

10 Form I. However, it was considered that a repeat of the experiment was guaranteed, but with less time elapsed after the formation of the precipitated oil. This again formed two transparent layers and resulted, gradually, a precipitated DMSO layer that was decanted and analyzed both wet and dry (CG1110Awet and CG1110A2 respectively). The DMSO liquor was allowed to reduce to a solid which was dried under vacuum (CG1110B2). The results shown in Figure 5 showed that all samples were finally

15 identical to the starting material (CG1099SM).

Example 4. Saturated Solutions

Saturated solutions are by far the best method for obtaining new polymorphs. Therefore, it

20 dissolved known amounts of Form I of RPL554 or were suspended over time to obtain a saturated solution at temperature, followed by polishing filtration to remove any form of memory (seminal). Subsequently, it was used in controlled cooling to promote crystallization.

Experimental (JN376):

25 Solvents were stirred at 45 ° C in the presence of 30 mg of Form I of RPL554 to leave an initial suspension. The suspensions were heated at 45 ° C for a period of 4.5 hours before hot filtration inside pre-heated tubes which were then allowed to evaporate slowly in front of the vacuum drying at 45 ° C and analysis. Note that inputs A and B (from Table 3) were heated at 35 ° C under nitrogen to help

30 evaporation for 24 hours.

Table 3. Solvent listings and results for saturated dissolution experiments (JN376)

Solvent

Grid Entry Observation XRPD

Tetraline

A1 Soln  rubber / oil n / a

NMP

A2 thin XT growth I form

Ethylene glycol

A3 insol n / a

DMSO

A4 XT thick growth New DSC Form collected

Anisole

TO 5 insol / trace oil n / a

DMF

A6 XT plus oil, oven dried I form

Cumene

B1 insol n / a

3-Me-1-BuOH

B2 Powder / rubber, oven dried Insufficient

n-BuoAc

B3 insol / trace oil n / a

Toluene

B4 insol / trace oil n / a

Dioxane

B5 trace glass, oven dried I form

Water

B6 insol n / a

Heptane

C1 insol n / a

n-PrOH

C2 trace dust Insufficient

IPA

C3 trace dust I form

CH3CN

C4 XT fine I form

MEK

C5 trace dust Insufficient

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Solvent

Grid Entry Observation XRPD

EtOH

C6 trace dust n / a

EtOAc

D1 insol n / a

THF

D2 insol n / a

MeOH

D3 insol n / a

Acetone

D4 insol n / a

TBME

D5 insol n / a

DCM

D6 powder I form

Key: XT = crystalline, insol = insoluble / very little material, trace dust -insufficient = not enough material to analyze

From the table it can be seen that many of the experiments do not dissolve a sufficient amount of material to allow analysis and returned either an oil stain or powder on the bottom of the tube after drying. However, despite this, a number of experiments returned the material to the form of

5 powder that allowed the collection of the XRPD analysis. The most satisfactory experiments were collected, which had shown the proliferation of formal crystals from maternal liquors, and only one entry showed any change of relevant form (DMSO).

The results of the DMSO input are shown below (Figures 6 and 7) and although the material shows

10 some similarity with Form I, it is thought that a new polymorph is present, based on XRPD and allowing a preferred orientation. The DSC analysis of the material provides a clear indication of the change in shape with a much smaller appearance of the main endotherm. There was insufficient material for the TGA and NMR analysis. According to the inventors' point of view, this is a less stable form of RPL554.

15 Example 5. Suspension maturations

24 suspension maturations were carried out in an attempt to force the current crystalline solid to undergo a shape transformation. The most effective method for this issue is to use prolonged heat / cold cycles, as submitted to Form I of RPL554.

20 Experimental (CG1100):

20 mg of material was stirred in glass tubes in approximately 30 volumes of solvent and cycled between isothermal periods (8 hours) of 20 ° C and 50 ° C. After 4 days of cycling, the solids were filtered and

25 dried under vacuum at 45 ° C before analysis by means of XRPD. All results were compared with the starting material, RPL554 (Form I); see Table 4.

Table 4. Suspension maturation experimentation for RPL554 (CG1100)

Solvent

Grid Entry Observation XRPD

Tetraline

A1 Soln  evap slow Rubber / oil

NMP

A2 Soln  evap slow Rubber / oil

Ethylene glycol

A3 gummy solid n / a

DMSO

A4 powder I form

Anisole

TO 5 powder I form

DMF

A6 powder I form

Cumene

B1 Soln  evap slow Rubber / oil

3-Me-1-BuOH

B2 powder I form

n-BuoAc

B3 powder I form

Toluene

B4 powder I form

Dioxane

B5 powder I form

Water

B6 powder I form

Heptane

C1 powder I form

n-PrOH

C2 powder I form

IPA

C3 powder I form

CH3CN

C4 powder I form

MEK

C5 powder I form

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Solvent

Grid Entry Observation XRPD

EtOH

C6 powder I form

EtOAc

D1 powder I form

THF

D2 powder I form

MeOH

D3 powder I form

Acetone

D4 powder I form

TBME

D5 powder I form

DCM

D6 powder I form

All samples showed the same XRPD profile when compared to the starting material, demonstrating that Form I of RPL554 was extremely flexible to modify and most likely was the thermodynamically more stable form of the compound (Figure 8).

5

Example 6. Re-crystallizations of mixed solvent

Because the solubility profiles can be greatly modified by the presence of mixed solvent systems and these trends can be very difficult to predict, a series of re-plans were planned.

10 crystallizations using a variety of systems. This was considered another appropriate way to induce change in a way that leads to the discovery of new polymorphs.

During the studies, it is appreciated that dioxane and cumene (other than normal DMF, DMSO and Tetralin), show a degree of efficacy when dissolving RPL554. These solvents formed the basis of certain tests

15 initials that adopted polished filtered solutions of each solvent and RPL554 and subsequently hot mixed with a range of known anti-solvents of various types, maintaining a solution and subsequently allowing it to cool slowly.

Experimental (PF87):

20 Stock solutions with 100 mg of RPL 554 were prepared by stirring the solid in 5 ml of the main solvent and allowing to warm to 50 ° C to improve the solution. An extra 1 ml (more if necessary) of co-solvent was added to improve the solution and form the final stock mixture. Samples were allowed to stir at 50 ° C for 30 minutes. On a preheated hot tube, (50 ° C), the solution of

Reserve followed by 2 ml of the anti-solvent shown in the table; The samples were allowed to stir and evaporate. The samples were filtered and dried overnight in a vacuum oven at 50 ° C in front of the analysis by means of XRPD (see Table 5).

Table 5. Mixed solvent crystallization of the reserve co-solvents indicated with the addition of anti-solvent 30 (PF87)

PF87

Anti-solvents 2-butanone EtOAc Heptane IPA MTBE

Reserve dissolution systems

Dioxane / DMSO (51)

I form I form I form I form I form I form

Dioxane / DMSO (5/2)

I form I form I form I form I form I form

2MeTHF / DMSO (5/1)

I form I form I form I form I form I form

DCM / Dioxane (5/1)

I form I form I form I form I form I form

All the experiments listed in the PF87 experiment returned Form I, which acts as additional proof of the thermodynamic stability of RPL554.

35 Example 7. Crystallization tests in mixed aqueous systems.

A series of mixed solvent crystallizations having an aqueous base in the PF86 experiment was investigated.

Experimental (PF86):

A hot stock solution of RPL554 (Form I) in DMSO (1 ml, 15 mg.ml-1) was added to a hot solvent solution, mixed with water (1: 1, v / v, 1 ml) as indicated in Table 6 (50 ° C). The solutions were allowed to stir at temperature for 48 hours, cooled, filtered and subsequently dried in the oven overnight (40 ° C). The XRPD analysis was carried out and compared with the starting material.

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Table 6. Summary of the results of the mixed aqueous crystallizations (experiment PF86)

Solvent system (+ water)

Expt XRPD

Acetonitrile

PF86A Identical to Form I

Dioxane

PF86B Identical to Form I

Ethanol

PF86C Identical to Form I

IPA

PF86D Identical to Form I

MeOH

PF86E Identical to Form I

THF

PF86F Identical to Form I

The results for this series of experiments (Figure 9) again showed that Form I was dominant.

5 Example 8. Generation of amorphous material

Because there was a historical experimental test of amorphous material generation by heating Form I of RPL554 to 245 ° C and rapid melting of the melt, a repetition of this method (TGA base) was carried out and produced same transparent material, yellow vitreous type.

10 Subsequently, this test sample was analyzed by means of XRPD and it was shown to be amorphous (see Figure 10).

Subsequently, this material was processed on the DSC instrument in an attempt to observe the glass transition and, if possible, any re-crystallizations. Figure 11 shows the results.

15 A proposed glass transition can be observed at approximately 120 ° C, with no formal re-crystallization observed. Although this experiment was operative, the sample was sent to LC analysis, since it is known that temperature stability is a potential issue (TGA observation). The chromatogram showed the presence of multiple peaks with only 26% of the material that was the starting material, RPL554. This result indicated

20 that it was either necessary to carry out the crushing cooling with more sensitive experimental margins or the method was invalid. The experiment was repeated at a low temperature (240 ° C), thus trying to avoid decomposition and, thus, return a better profile in terms of purity. The LC analysis returned a similar profile of very low purity and several secondary products. Therefore, it was evident that, in this way, the amorphous material would not be generated.

Subsequently, a number of other attempts were made to generate the amorphous phase, beginning with physical manipulation (grinding, applied pressure) and subsequently rapid evaporation of the diluted solution from solvents such as DCM and cumene. In this experiment, it was shown that the ground sample (mortar and grinder) was very electrostatic and failed to transfer to the Kapton plate for XRPD analysis. Do not

However, the sample was successfully transferred to a DSC cup; the melting point observed was lower than that of the reference material, indicating the possibility of changing shape. However, a repeat of this experiment is required for this additional investigation; however, the amorphous phase was not the next one based on this approach (Figure 12).

35 Applied pressure (100 KN): 10 mg of RPL554 was left in a pressure disc system overnight, forming a thin and uniform disk with a thickness of less than 0.1 mm. The disc was ground uniformly and analyzed by means of XRPD (Figure 13).

The comparison of XRPD of the pressurized material with the Starting Form (CG1099F reference used) demonstrates

40 that the compression of the crystalline material generates an amorphous phase. Following these observations, a thermal analysis (DSC) was carried out and the material was shown to be amorphous (Figure 14).

The transition from the amorphous material is shown by means of Tg at 75 ° C, which subsequently recrystallizes at approximately 131 ° C before melting at 242.6 ° C and 262.7 ° C, consecutively. The first endotherm

It appears to be slightly lower than the reference material and could potentially be related to a different form (purity comparisons would be a sensitive study to carry out before assigning a new form). The second melt could also be considered as a different crystalline entity, although it should be emphasized that it is in the region of decomposition for this material.

Rapid evaporation of solvent: a flask containing RPL554-070638 was dissolved completely in a large volume of DCM at room temperature (290 ml, 2.0 g). Subsequently, the solvent was removed quickly at temperature, keeping RPL554-070638 completely dissolved, avoiding a slow precipitation of the raw material and the presence of Form I. The HPLC analysis showed a high chemical purity and proton NMR indicated a residual DCM trace.

XRPD of the initial batch showed an amorphous phase (JN367E), which was confirmed by means of DSC (Figure 15). The process

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heating induces a large exotherm, which illustrates a crystallization at 152 ° C, followed by an endotherm for the expected melt of Form I, although slightly suppressed. Note the small differences between this batch and the amorphous material derived from the applied pressure (the main difference in the temperature of the initial exotherm and the profile after the main fusion).

5 Having successfully isolated a small amount of amorphous material, this technique was scaled to work in terms of grams of material for use in the saturated solution and suspension. Figure 16 shows the reference XRPD profile for this lot (JN376F).

10 Example 9. Saturated solutions with amorphous material

As indicated, saturated solutions are the best method of obtaining pure phases of new polymorphs. For this, the amorphous material was taken and a study was carried out using saturated solutions, as in JN376, in order to obtain a high total starting concentration.

15 Experimental (PF89):

24 solvents were stirred at 45 ° C in the presence of 30 mg of RPL554 (JN76F) to leave an initial suspension (more concentrated) than with the crystalline phase. The suspensions were heated for a period of 4.5 hours to

20 increase solubility. Each tube was checked for complete dissolution of the material. If they remained solid, an extra 1 ml of the known solvent was added until it completely dissolved. Each tube was evaluated before hot filtration in pre-heated tubes that were subsequently allowed to evaporate slowly. Note that the tubes were placed under nitrogen to contribute to evaporation for 24 hours. The following table (Table 7) shows the results of this experiment.

25 Table 7. Saturated dissolution experiments (amorphous phase input, expt PF89)

Solvent

Grid Entry Observation XRPD

Tetraline

A1 Oil N / A

NMP

A2 Not dry Wet but Form I

Ethylene glycol

A3 No precipitation was observed N / A

DMSO

A4 Liquid + crystals Mixed form (measured wet)

Anisole

TO 5 Rubber / oil N / A

DMF

A6 Liquid + crystals I form

Cumene

B1 Liquid Insufficient mesh

3-Me-1-BuOH

B2 Crystals I form

n-BuoAc

B3 Crystals I form

Toluene

B4 Crystals I form

Dioxane

B5 Crystals I form

Water

B6 Insol N / A

Heptane

C1 Solid I form

n-PrOH

C2 Solid I form

IPA

C3 Solid I form

CH3CN

C4 Extra peaks I form

MEK

C5 Extra peaks I form

EtOH

C6 Solid I form

EtOAc

D1 Solid I form

THF

D2 Extra peaks I form

MeOH

D3 1st peak separation I form

Acetone

D4 - Insufficient mesh

TBME

D5 Amorphous N / A

DCM

D6 Amorphous N / A

From the table, it can be seen that some experiments dissolved an insufficient amount of material to allow analysis, or returned a spot of amorphous oil or powder on the bottom of the tube 30 after drying. However, despite this, a number of experiments returned material in powder form that allowed the collection of the XRPD analysis. They have collected the most satisfactory experiments, showing

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some formal crystals in development from maternal liquors, and only one entry showed some change of relevant form (PF89A4, DMSO).

The results for DMSO input are shown below, and while the material shows some similarity

5 with Form I, it is thought that a new polymorph is present based on XRPD and allows a preferred orientation. The DSC analysis of the material provides a clear indication of the change in shape with a much lower appearance of the main endotherm. There was insufficient material for the TGA and NMR analysis. This result was scaled for further investigation, although it is appreciated that this material did not present the same profile, but rather a relative profile altered with respect to RPL554 as JN376A4 (saturated solution from DMSO, Figures 6 and 7). Be

10 considered that this was due to the relative level of solvation.

In order to contribute to the understanding of the described analysis, and due to the fact that high quality modeling information had been obtained from the predicted unit cell parameters (indexing experiment) for RPL554, the new phase was compared with This theoretical data.

15 The numerical analysis shows that most of the material is in form I with some spikes that do not fit. This indicated strong evidence that the sample was a mixture of two forms, one potentially a solvated product. The results have not been fully presented and were conclusive in that Form I was observed in all cases in which there was sufficient material for collection, except for DMSO, in which

20 case there was a small change in the XRPD profile.

Example 10. Automated experiment (expanded anti-solvent study)

An experiment was initiated to extend the solvent range from which it was used in the work of adding

25 anti-solvent and to carry out said solvent mixture at temperature, instead of crushing cooling by using anti-solvent (CG1099).

Experimental (PF85):

A defined amount of RPL554 (1.5 g) and 20 ml of DMSO was added to a tube heated at 50 ° C to improve dissolution. 200 µl of stock solution was placed in a hot well (50 ° C). 200 µl of the listed solvent (following table) was added to the well and allowed to stir for 72 hours before isolation. The samples were subsequently transferred onto a Kapton plate for XRPD analysis. The results were processed and compared with the starting material RPL554 (Table 8). When XRPD was insufficient to

35 differentiated from the starting material, thermal analysis was carried out in order to define the melting points (material that is left).

Table 8. Automated crystallizations of hot mixed solvent (PF85), detailing the initial results by means of XRPD and DSC when possible

Solvent

Grid Entry DSC (appearance / peak) Form I ref. = (246/248) XRPD conclusion Total conclusion (DSC / XRPD)

Formamide

A12 Wet sample I form I form

N-methyl-2-pyrrolidinone (NMP)

A11 241 ° C / 247 ° C Poor diffraction (probably Form I) I form

Ethylene glycol

A10 Rubber I form I form

Dimethyl Sulfoxide (DMSO)

A9 243 ° C / 248 ° C Form I + extra peaks I form

N, N-dimethylacetamide

A8 Form I + extra peaks I form

Anisole

A7 Form I + extra peaks I form

N, N-dimethylformamide

A6 Form I + extra peaks I form

Cumene

TO 5 Rubber 240 ºC / 246.2 ºC Different from the starting material Form II

1-pentanol

A4 Poor diffraction (probably Form I) I form

Chlorobenzene

A3 241 ° C / 246.7 ° C Slightly different Form II

3-methyl-1-butanol

A2 I form I form

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Solvent

Grid Entry DSC (appearance / peak) Form I ref. = (246/248) XRPD conclusion Total conclusion (DSC / XRPD)

Methylbutyl ketone = 2hexanone

A1 I form I form

Acetate of butilo

B12 Rubber I form I form

2-methoxyethanol

B11 I form I form

Acetic acid

B10 241.6 ° C / 247.4 ° C Strongly preferred orientation Form II

1-Butanol

B9 Rubber Wet sample but Form I I form

Methyl Isobutyl Ketone = 4 Methyl 2 Pentanone

B8 I form I form

Isobutyl acetate

B7 I form I form

Pyridine

B6 I form I form

Toluene

B5 239.5 ° C / 246 ° C Preferred orientation (most likely Form I) Form II

2-Methyl-1-propanol

B4 233 ° C / 243 ° C Preferred orientation Form III

Propylacetate

B3 Form I + extra peaks I form

Nitromethane

B2 Form I + extra peaks I form

Dioxane

B1 243 ° C / 248 ° C Uncertain due to preferred orientation I form

Methylcyclohexane

C12 I form I form

Formic acid

C11 I form I form

Water

C10 243 ° C / 247 ° C I form

2-Butanol

C9 I form I form

Heptanos

C8 I form I form

1-Propanol

C7 242 ° C / 247 ° C I form I form

Isopropyl acetate

C6 I form

1,2-Dimethoxy-ethane (glyme, DME)

C5 242.3 ° C / 248 ° C I form

2-Propanol

C4 I form I form

Acetonitrile

C3 I form I form

Cyclohexane

C2 I form I form

Ethanol

C1 I form I form

Ethyl acetate

D12 Rubber. Two endotherms, 226 ° C / 268 ° C PO or different 2 endoses, potentially Form IV and V

1,1,2-Trichloroethene

D11 Form I + extra peaks I form

Hexane

D10 223.6 ° C Preferred orientation Pure IV form

Tetrahydrofuran (THF)

D9 Rubber Preferred guidance for Form I I form

Methanol

D8 242 ° C / 247 ° C Different or Form I (PO?) I form

Chloroform

D7 Form I + extra peaks I form

Methyl acetate

D6 I form I form

Acetone

D5 I form I form

Tert-Butylmethyl ether (MTBE)

D4 Form I + extra peaks I form

Ethyl formate

D3 I form I form

Dichloromethane

D2 I form I form

1,2-Dichloroethene

D1 Form I + extra peaks I form

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It can be seen from the tabulated results, especially those indicated by the traces collected from DSC, that certain mixed or potentially new phases were isolated from the broader anti-solvent library. Although XRPD patterns may appear initially different, due to peak intensities, preferred orientations may be present and thus further analysis may be necessary in

5 depth (preferred orientation was considered possible due to visual observation of the crystals transferred from the automated experiment) (results not shown). This analysis was also presented by means of numerical comparison of the new XRPD patterns with the predicted pattern of Form I from the indexed. The idea was to gain a lot of quality data on the presence of a certain new phase or if the result was, in fact, a mixture from the initial samples.

10 For the PF85 experiment, the results provided some relatively interesting information about the ability of RPL554 to exhibit different crystalline forms. The combined analytical tools (XRPD / DSC) revealed that there were potentially at least five crystalline forms along Form I of RPL554, although the feeling was that these were mostly mixtures. The experiments that returned the evidence

15 most likely of new phases and were selected for scaling were (note that other systems can replicate these results):

PF85A3 Chlorobenzene / DMSO PF85A5 Cumene / DMSO PF85B4 1-Methyl-2-propanol / DMSO PF85D10 Hexane / DMSO PF8512 Ethyl acetate / DMSO

Numerical comparison of the previous 5 forms with the calculated Form I pattern of RPL554.

20 Data referring to the new potential polymorphs of RPL554 and the calculated profile of Form I (data not shown). For this study, reference is made to the unit cell parameters found while trying to index Form I of RPL554. The algorithm in the software allows the entry of unit cell parameters, in this case those related to Form I. Subsequently, the software calculates a profile, allowing

25 thus the user visually compare with the measured data. This approach contributes to the confirmation of the existence of a new form and / or a new form that coexists with Form I (mixture). In almost all the previous cases, the appreciated results suggest that Form I was present and there have been mainly mixtures instead of a new pure phase, with the potential for D10 to be the exception. All previous candidates were chosen for escalation.

30

Example 11. Scaling of potential new forms

A series of scaling experiments were initiated in order to generate more material in new and / or mixed forms. In this way, a detailed study could be carried out and crossing suspensions could be attempted. Should

35 it can be appreciated that such scaling may not be trivial, especially when less stable forms have been induced on small scales (the subtleties of the process mean that the scaling may be being tested).

Experimental (PF90 and JN386):

A stock solution of RPL554 in hot DMSO was prepared. The solution was filtered with polishing and 1.75 ml (approximately 200 mg of content) was added to a volume (4 ml) of hot solvent (55 ° C).

Samples were left under stirring and slow evaporation over time in order to simulate the conditions that

45 generated the new potential polymorphs. After precipitation, the samples were filtered and dried in vacuo at 40 ° C before analysis by means of XRPD. The following table (Table 9) collects the scaling experiments for RPL554.

Table 9. Results for the scaling of potential polymorphs showing the predominance of Form I

Solvents (DMSO and ...)

Observations XRPD

Ethyl Acetate (JN3 84A)

Precipitated in powder form, in 24 hours I form

2-Me-1-Propanol PF90

Fine crystals I form

PF90 acetic acid

Rubber N / A

Chlorobenzene PF90

A small part of the wet sample was placed on XRD I form

Pumene PF90

Very fine powder I form

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Solvents (DMSO and ...)

Observations XRPD

DMSO PF90

A solid formed with decent sized crystals XRPD slightly different (see below)

Hexane PF90

The sample was allowed to evaporate slowly without stirring. I form

With the materials at hand, the analysis such as TGA would be applicable to determine if solvation is the cause of the differences in XRPD profiles (for example, the mixed phase of Form 1 and a solvate), or if It is due to the mixture of non-solvated forms. In the case of ethyl acetate, for example, it can be seen that the scaling 5 of the process returns Form 1 and, thus, most likely the most marked differences in the original XRPD profile are due to a mixture of Form 1 and a less stable form that has become the most stable polymorph (the low pf endotherm present in DSC, not within the decomposition phase is supportive in this matter). The same content is true for the other solvents except acetic acid, which returned a gum and DMSO, which returned a mixture. In this case, there was enough material available for

10 confirm the presence of Form I and a less stable form which, over time, becomes the most stable form. It should be appreciated that this is not identical to the less stable forms identified from DMSO in PF98A4 and JN376A4, showing that this solvent provides a number of changes to the main form, but that the scaling of Form I was still the main component.

15 The analysis (Figures 18 and 19) is related to the scaling experiment involving DMSO. The XRPD data again shows the presence of peaks that do not belong to Form I. As demonstrated in the thermal analysis, Form I is present in the majority (melting start at 246.1 ° C), while the coexistence of presence of a high melting compound (start at 261 ° C). Gravimetric analysis demonstrates the presence of free DMSO in the sample, which evaporates under a stream of nitrogen in TGA when

20 causes heating of the sample from room temperature to 100 ° C. This cannot be related to the inclusion of the solvent in the high melting compound. Although it seems that all free DMSO disappears after heating, the TGA curve shows a slow but steady weight loss between 100 ° C and 200 ° C that may be related to "included" DMSO, although without isolation of a pure phase, this assumption It is difficult to assume. Proton NMR, as expected, shows the presence of DMSO in the sample.

25 By way of conclusion, the scaling experiments for RPL554 demonstrate that Form I remains privileged and also kinetically more favored. It is likely that most of the observed mixed phases will eventually become Form I.

30 Example 12. Aqueous solubility

Because the amorphous material and Form I were at hand, both samples were exposed to a direct study of aqueous solubility in purified water at 25 ° C. The experiment was carried out since the initial isolation of the crystalline or amorphous material during a previous synthesis campaign was unknown.

35 Experimental:

A 20 mg suspension of the candidate was mixed in purified water (1 ml, pH 7). The suspension was equilibrated at 25 ° C for 24 hours. The suspension was then filtered in an HPLC vessel and then the filtrate was diluted by means of an appropriate factor, the quantification being carried out by means of HPLC with reference to a standard solution.

Outcome:

Amorphous JN376F 0.03 mg / ml RPL554 Form I 0.001 mg / ml

45 As expected, it was shown that the amorphous phase, although quite insoluble, was more soluble than Form I in this basic test. Unfortunately, both samples were too fine for effective filtration and isolation for re-analysis by means of XRPD, although it is known that Form I comes from such suspensions.

50 Example 13. X-ray data. Relevant parameters related to the individual crystal RPL554

The X-ray data for RPL554 can be found in Tables 10-15, and Figures 20-22.

Table 10. Glass and refined structure data for RPL554 (onyx2010a). Identification code onyx2010a Empirical formula C26 H31 N5 O4 Formula weight 477.56

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Temperature 123 (2) K Wavelength 0.71073 angstrom Triclinic crystal system Space group P-1 Unit cell dimensions a = 8.1246 (4) angstrom  = 91.583 (4) º b = 11.4573 (5) angstrom  = 90,299 (4) º c = 13,2398 (6) angstrom  = 99,628 (4) º Volume 1214.56 (10) angstrom3 Z2 Density (calculated) 1,306 Mg / m3 Absorption coefficient 0.090 mm-1 F ( 000) 508 Glass size 0.32 x 0.22 x 0.08 mm3 Theta interval for data collection 2.86 to 30.72º Index intervals -11 <= h <= 11, -16 <= k < = 16, -17 <= 1 <= 7 Reflections collected 13256 Independent reflections 6619 [R (int) = 0.0281] Fullness up to theta = 27.50 º 99.9% Semi-empirical absorption correction from equivalents Transmission max. and min. 1,00000 and 0.94889 Refining method Full matrix least squares on F2 Data / limitations / parameters 6619/0/333 Goodness of fit on F2 0.854 Final R indices [I> 2 sigma (I)] R1 = 0.0420 , wR2 = 0.0852 R indices (all data) R1 = 0.0802, wR2 = 0.0913 Higher peak and valley difference 0.309 and -0.240 and angstrom-3

Table 11. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (angstrom2 x 103) for onyx2010a. U (eq) is defined as one third of the trace of the tense Uij turned orthogonal. x and z U (eq)

O (1)

2377 (1) 8343 (1) 861 (1) 21 (1)

O (2)

-7077 (1) 8128 (1) 3364 (1) 22 (1)

O (3)

-7605 (1) 5925 (1) 2796 (1) 22 (1)

O (4)

3558 (1) 13706 (1) 485 (1) 24 (1)

N (1)

-69 (1) 8094 (1) 1717 (1) 16 (1)

N (2)

1803 (1) 9907 (1) 1810 (1) 16 (1)

N (3)

1201 (1) 11536 (1) 2713 (1) 18 (1)

N (4)

4497 (2) 11982 (1) 120 (1) 21 (1)

N (5)

6214 (2) 13771 (1) -66 (1) 21 (1)

C (1)

1430 (2) 8757 (1) 1440 (1) 16 (1)

C (2)

742 (2) 10441 (1) 2447 (1) 16 (1)

C (3)

-772 (2) 9687 (1) 2707 (1) 17 (1)

C (4)

-1198 (2) 8572 (1) 2327 (1) 15 (1)

C (5)

-2840 (2) 7838 (1) 2490 (1) 15 (1)

C (6)

-3091 (2) 6628 (1) 2273 (1) 16 (1)

C (7)

-1613 (2) 6080 (1) 1962 (1) 20 (1)

C (8)

-487 (2) 6879 (1) 1271 (1) 20 (1)

C (9)

-4174 (2) 8364 (1) 2861 (1) 17 (1)

C (10)

-5720 (2) 7695 (1) 2993 (1) 17 (1)

C (11)

-6002 (2) 6483 (1) 2709 (1) 17 (1)

C (12)

-4684 (2) 5960 (1) 2371 (1) 18 (1)

C (13)

-6739 (2) 9152 (1) 4018 (1) 26 (1)

C (14)

-7922 (2) 4669 (1) 2626 (1) 27 (1)

C (15)

16 (2) 12112 (1) 3230 (1) 17 (1)

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x

Y z U (eq)

C (16)

318 (2) 12495 (1) 4241 (1) 20 (1)

C (17)

-752 (2) 13173 (1) 4697 (1) 23 (1)

C (18)

-2120 (2) 13474 (1) 4191 (1) 23 (1)

C (19)

-2386 (2) 13077 (1) 3191 (1) 22 (1)

C (20)

-1340 (2) 12405 (1) 2698 (1) 19 (1)

C (21)

1802 (2) 12195 (1) 4804 (1) 31 (1)

C (22)

-3260 (2) 14224 (1) 4688 (1) 34 (1)

C (23)

-1614 (2) 12080 (1) 1592 (1) 29 (1)

C (24)

3339 (2) 10637 (1) 1453 (1) 18 (1)

C (25)

3023 (2) 11218 (1) 464 (1) 20 (1)

C (26)

4689 (2) 13185 (1) 189 (1) 17 (1)

Table 12. Link lengths [angstrom] and angles [º] for onyx2010a. O (1) -C (1) 1,2296 (13) O (2) -C (10) 1,3707 (14) O (2) -C (13) 1,4268 (15) O (3) - C (11) 1,3583 (15) O (3) -C (14) 1.4299 (16) O (4) -C (26) 1,2363 (14) N (1) -C (1) 1 , 3803 (16) N (1) -C (4) 1.3948 (14) N (1) -C (8) 1.4812 (16) N (2) -C (1) 1.3779 (16) N (2) -C (2) 1.4084 (15) N (2) -C (24) 1.4686 (16) N (3) -C (2) 1.2858 (16) N (3) - C (15) 1.4240 (15) N (4) -C (26) w1.3610 (17) N (4) -C (25) 1.4436 (17) N (4) -H (1N) 0.847 (14) N (5) -C (26) 1,3558 (17) N (5) -H (3N) 0.897 (16) N (5) -H (2N) 0.876 (15) C (2) -C (3) 1,4300 (18) C (3) -C (4) 1,3488 (17) C (3) -H (3) 0.9500 C (4) -C (5) 1.4735 (17 ) C (5) -C (6) 1,3896 (17) C (5) -C (9) 1.4093 (16) C (6) -C (12) 1.3971 (18) C (6) -C (7) 1,4998 (17) C (7) -C (8) 1,5121 (18) C (7) -H (7A) 0.9900 C (7) -H (7B) 0.9900 C (8) -H (8A) 0.9900 C (8) -H (8B) 0.9900 C (9) -C (10) 1.3725 (18) C (9) -H (9) 0, 9500 C (10) -C (11) 1.4099 (18) C (11) -C (12) 1.3826 (17) C (12) -H (12) 0.9500 C (13) -H ( 13A) 0.9800 C (13) -H (13B) 0.9800 C (13) -H (13C) 0.9800

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C (14) -H (14A)

0.9800

C (14) -H (14B)

0.9800

C (14) -H (14C)

0.9800

C (15) -C (20)

1.3972 (18)

C (15) -C (16)

1.4038 (17)

C (16) -C (17)

1,3884 (17)

C (16) -C (21)

1,5074 (19)

C (17) -C (18)

1.3923 (19)

C (17) -H (17)

0.9500

C (18) -C (19)

1.3918 (18)

C (18) -C (22)

1,5072 (17)

C (19) -C (20)

1.3914 (17)

C (19) -H (19)

0.9500

C (20) -C (23)

1.5068 (18)

C (21) -H (21A)

0.9800

C (21) -H (21B)

0.9800

C (21) -H (21C)

0.9800

C (22) -H (22A)

0.9800

C (22) -H (22B)

0.9800

C (22) -H (22C)

0.9800

C (23) -H (23A)

0.9800

C (23) -H (23B)

0.9800

C (23) -H (23C)

0.9800

C (24) -C (25)

1,5258 (18)

C (24) -H (24A)

0.9900

C (24) -H (24B)

0.9900

C (25) -H (25A)

0.9900

C (25) -H (25B)

0.9900

C (10) -O (2) -C (13)

116.55 (10)

C (11) -O (3) -C (14)

117.16 (10)

C (1) -N (1) -C (4)

121.65 (11)

C (1) -N (1) -C (8)

116.95 (10)

C (4) -N (1) -C (8)

121.24 (11)

C (1) -N (2) -C (2)

123.78 (11)

C (1) -N (2) -C (24)

117.51 (10)

C (2) -N (2) -C (24)

118.54 (11)

C (2) -N (3) -C (15)

117.37 (11)

C (26) -N (4) -C (25)

122.90 (11)

C (26) -N (4) -H (1N)

118.4 (11)

C (25) -N (4) -H (1N)

118.7 (10)

C (26) -N (5) -H (3N)

116.1 (10)

C (26) -N (5) -H (2N)

118.0 (10)

H (3N) -N (S) -H (2N)

117.6 (14)

O (1) -C (1) -N (2)

121.24 (12)

O (1) -C (1) -N (1)

121.00 (12)

N (2) -C (1) -N (1)

117.73 (10)

N (3) -C (2) -N (2)

118.03 (12)

N (3) -C (2) -C (3)

127.18 (11)

N (2) -C (2) -C (3)

114.78 (11)

C (4) -C (3) -C (2)

122.70 (11)

C (4) -C (3) -H (3)

118.7

C (2) -C (3) -H (3)

118.7

C (3) -C (4) -N (1)

119.19 (12)

C (3) -C (4) -C (5)

123.14 (11)

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N (1) -C (4) -C (5) 117.61 (11) C (6) -C (5) -C (9) 119.51 (12) C (6) -C (5) - C (4) 120.45 (11) C (9) -C (5) -C (4) 120.04 (11) C (5) -C (6) -C (12) 119.59 (11) C (5) -C (6) -C (7) 118.11 (12) C (12) -C (6) -C (7) 122.29 (12) C (6) -C (7) - C (8) 111.05 (11) C (6) -C (7) -H (7A) 109.4 C (8) -C (7) -H (7A) 109.4 C (6) -C (7) -H (7B) 109.4 C (8) -C (7) -H (7B) 109.4 H (7A) -C (7) -H (7B) 108.0 N (1) - C (8) -C (7) 110.91 (10) N (1) -C (8) -H (8A) 109.5 C (7) -C (8) -H (8A) 109.5 N (1) -C (8) -H (8B) 109.5 C (7) -C (8) -H (8B) 109.5 H (8A) -C (8) -H (8B) 108.0 C (10) -C (9) -C (5) 120.58 (12) C (10) -C (9) -H (9) 119.7 C (5) -C (9) -H (9 ) 119.7 O (2) -C (10) -C (9) 124.53 (12) O (2) -C (10) -C (11) 115.61 (12) C (9) -C (10) -C (11) 119.83 (11) O (3) -C (11) -C (12) 125.46 (12) O (3) -C (11) -C (10) 115, 06 (11) C (12) -C (11) -C (10) 119.48 (12) C (11) -C (12) -C (6) 120.80 (12) C (11) -C (12) -H (12) 119.6 C (6) -C (12) -H (12) 119.6 O (2) -C (13) -H (13A) 109.5 O (2) - C (13) -H (13B) 109.5 H (13A) -C (13) -H (13B) 109.5 O (2) -C (13) -H (13C) 109.5 H (13A) -C (13) -H (13C) 109.5 H (13B) -C (13) -H (13C) 109.5 O (3) -C (14) -H (14A) 109.5 O (3 ) -C (14) -H (14B) 109.5 H (14A) -C (14) -H (14B) 109.5 O (3) - C (14) -H (14C) 109.5 H (14A) -C (14) -H (14C) 109.5 H (14B) -C (14) -H (14C) 109.5 C (20) -C (15) -C (16) 120.45 (11) C (20) -C (15) -N (3) 119.62 (11) C (16) -C (15) -N (3) 119.56 (12) C (17) -C (16) -C (15) 118.75 (12) C (17) -C (16) -C (21) 121.09 (12) C (15) -C (16) -C (21) 120.15 (11) C (16) -C (17) -C (18) 122.27 (12) C (16) -C (17) -H (17) 118.9 C (18) -C (17) -H (17) 118.9 C (19) -C (18) -C (17) 117.49 (12) C (19) -C (18) - C (22) 120.62 (13) C (17) -C (18) -C (22) 121.87 (12)

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C (20) -C (19) -C (18) 122.35 (13) C (20) -C (19) -H (19) 118.8 C (18) -C (19) -H (19 ) 118.8 C (19) -C (20) -C (15) 118.69 (12) C (19) -C (20) -C (23) 119.92 (12) C (15) -C (20) -C (23) 121.26 (11) C (16) -C (21) -H (21A) 109.5 C (16) -C (21) -H (21B) 109.5 H ( 21A) -C (21) -H (21B) 109.5 C (16) -C (21) -H (21C) 109.5 H (21A) -C (21) -H (21C) 109.5 H (21B) -C (21) -H (21C) 109.5 C (18) -C (22) -H (22A) 109.5 C (18) -C (22) -H (22B) 109.5 H (22A) -C (22) -H (22B) 109.5 C (18) -C (22) -H (22C) 109.5 H (22A) -C (22) -H (22C) 109, 5 H (22B) -C (22) -H (22C) 109.5 C (20) -C (23) -H (23A) 109.5 C (20) -C (23) -H (23B) 109 , 5 H (23A) -C (23) -H (23B) 109.5 C (20) -C (23) -H (23C) 109.5 H (23A) -C (23) -H (23C) 109.5 H (23B) -C (23) -H (23C) 109.5 N (2) -C (24) -C (25) 110.92 (11) N (2) -C (24) - H (24A) 109.5 C (25) -C (24) -H (24A) 109.5 N (2) -C (24) -H (24B) 109.5 C (25) -C (24) -H (24B) 109.5 H (24-A) -C (24) -H (24B) 108.0 N (4) -C (25) -C (24) 111.97 (11) N (4 ) -C (25) -H (25A) 109.2 C (24) -C (25) -H (25A) 109.2 N (4) -C (25) -H (25B) 109.2 C ( 24) -C (25) -H (25B) 109.2 H (25A) -C (25) -H (25B) 107.9 O (4) -C (26) -N (5) 122.27 ( 13) O (4) -C (26) -N (4) 122.15 (13) N (5) -C (26) -N (4) 11 5.55 (12)

Table 13. Anisotropic displacement parameters (angstrom2 x 103) for onyx2010a. The exponent of the anisotropic displacement factor takes the form: -22 [h2a * 2U11 + ... + 2 h k a * b * U12]

U11 U22 U33

U23

U13 U12.

O (1) 19 (1) 20 (1) 24 (1) -1 (1) 6 (1) 4 (1) O (2) 16 (1) 20 (1) 28 (1) -4 (1) 4 (1) 3 (1) O (3) 16 (1) 16 (1) 32 (1) -1 (1) 2 (1) -2 (1) O (4) 23 (1) 20 (1) 30 (1) 1 (1) 8 (1) 7 (1) N (1) 15 (1) 14 (1) 19 (1) -1 (1) 2 (1) 2 (1) N (2) 14 (1) 14 (1) 19 (1) 1 (1) 4 (1) 0 (1) N (3) 17 (1) 16 (1) 19 (1) -1 (1) 1 (1) 1 ( 1) N (4) 20 (1) 16 (1) 28 (1) 3 (1) 10 (1) 3 (1) N (5) 20 (1) 15 (1) 28 (1) 3 (1) 6 (1) 2 (1) C (1) 16 (1) 16 (1) 17 (1) 2 (1) 0 (1) 3 (1) C (2) 16 (1) 16 (1) 16 ( 1) 2 (1) 0 (1) 3 (1) C (3) 15 (1) 16 (1) 19 (1) 0 (1) 3 (1) 2 (1)

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U11 U22

U33

U23

U13

U12

C (4) 14 (1) 16 (1) 16 (1) 3 (1) 1 (1) 3 (1) C (5) 15 (1) 15 (1) 14 (1) 1 (1) -1 (1) 1 (1) C (6) 16 (1) 15 (1) 17 (1) 1 (1) 0 (1) 2 (1) C (7) 19 (1) 14 (1) 27 (1 ) -1 (1) 1 (1) 2 (1) C (8) 19 (1) 14 (1) 25 (1) -4 (1) 3 (1) 1 (1) C (9) 18 (1 ) 14 (1) 19 (1) 0 (1) 1 (1) 1 (1) C (10) 17 (1) 19 (1) 16 (1) 2 (1) 1 (1) 5 (1) C (11) 14 (1) 18 (1) 19 (1) 3 (1) -1 (1) -1 (1) C (12) 20 (1) 13 (1) 20 (1) 0 (1) 0 (1) 0 (1) C (13) 24 (1) 24 (1) 32 (1) -6 (1) 4 (1) 6 (1) C (14) 22 (1) 17 (1) 39 ( 1) -1 (1) 7 (1) -5 (1) C (15) 16 (1) 12 (1) 21 (1) 0 (1) 3 (1) -2 (1) C (16) 23 (1) 16 (1) 21 (1) 2 (1) 1 (1) 1 (1) C (17) 31 (1) 21 (1) 17 (1) 0 (1) 3 (1) 3 (1 ) C (18) 26 (1) 19 (1) 24 (1) 2 (1) 8 (1) 4 (1) C (19) 20 (1) 20 (1) 26 (1) 3 (1) 0 (1) 3 (1) C (20) 20 (1) 14 (1) 21 (1) 0 (1) 0 (1) -1 (1) C (21) 37 (1) 34 (1) 22 ( 1) -3 (1) -7 (1) 11 (1) C (22) 38 (1) 38 (1) 30 (1) -2 (1) 7 (1) 16 (1) C (23) 33 (1) 34 (1) 23 (1) -4 (1) -7 (1) 13 (1) C (24) 13 (1) 16 (1) 23 (1) 1 (1) 3 (1) 0 (1) C (25) 17 (1) 18 (1) 24 (1) 3 (1) 3 (1) 0 (1) C (26) 20 (1) 17 (1) 15 (1) 1 (1 ) 1 (1) 2 (1)

Table 14. Hydrogen coordinates (x 104) and anisotropic displacement parameters (angstrom2 x103) for ony2010a. x and z U (eq)

H (3) -1508 9983 3166 20 H (7A) -2003 5309 1609 24 H (7B) -974 5932 2570 24 H (8A) 552 6554 1154 24 H (8B) -1053 6906 610 24 H (9) -4000 9189 3022 20 H (12) -4863 5137 2202 21 H (13A) -5908 9037 4528 40 H (13B) -7771 9279 4352 40 H (13C) -6305 9845 3621 40 H (14A) -7656 4481 1925 40 H ( 14B) -9101 4366 2750 40 H (14C) -7224 4300 3085 40 H (17) -542 13440 5379 28 H (19) -3315 13270 2832 26 H (21A) 1975 12671 5434 46 H (21B) 2798 12366 4385 46 H (21C) 1596 11352 4961 46 H (22A) -4074 13725 5103 51 H (22B) -3849 14592 4168 51 H (22C) -2599 14843 5117 51 H (23A) -2496 12476 1324 44

H (23B) -1945 11221 1508 44 H (23C) -577 12334 1225 44 H (24A) 4208 10135 1350 21 H (24B) 3754 11257 1972 21 H (25A) 2653 10595 -60 24 H (25B) 2114 11687 560 24 H (1N)

5292 (18) 11670 (13) -118 (11) 31 (4)

E11748611

07-21-2014 E11748611

X and Z

U (eq)

H (3N) H (2N)

6273 (19) 14542 (14) -193 (11) 6881 (19) 13378 (14) -400 (12) 31 (4) 36 (5)

Table 15: Torsion angles [º] for onyx2010a

C (2) -N (2) -C (1) -O (1) 177.74 (12)

C (24) -N (2) -C (1) -O (1) 2.58 (17)

C (2) -N (2) -C (1) -N (1) -0.33 (17)

C (24) -N (2) -C (1) -N (1) -175.49 (11)

C (4) -N (1) -C (1) -O (1) -176.36 (11)

C (8) -N (1) -C (1) -O (1) -1.08 (18)

C (4) -N (1) -C (1) -N (2) 1.72 (17)

C (8) -N (1) -C (1) -N (2) 177.00 (11)

C (15) -N (3) -C (2) -N (2) 170.96 (11)

C (15) -N (3) -C (2) -C (3) -7.54 (19)

C (1) -N (2) -C (2) -N (3) -177.51 (12)

C (24) -N (2) -C (2) -N (3) -2.40 (17)

C (1) -N (2) -C (2) -C (3) 1.18 (17)

C (24) -N (2) -C (2) -C (3) 176.30 (11)

N (3) -C (2) -C (3) -C (4) 174.92 (13)

N (2) -C (2) -C (3) -C (4) -3.63 (18)

C (2) -C (3) -C (4) -N (1) 5.10 (19)

C (2) -C (3) -C (4) -C (5) -172.25 (12)

C (1) -N (1) -C (4) -C (3) -4.08 (18)

C (8) -N (1) -C (4) -C (3) -179.16 (12)

C (1) -N (1) -C (4) -C (5) 173.41 (11)

C (8) -N (1) -C (4) -C (5) -1.67 (17)

C (3) -C (4) -C (5) -C (6) -166.10 (12)

N (1) -C (4) -C (5) -C (6) 16.51 (17)

C (3) -C (4) -C (5) -C (9) 14.28 (19)

N (1) -C (4) -C (5) -C (9) -163.11 (11)

C (9) -C (5) -C (6) -C (12) 3.69 (18)

C (4) -C (5) -C (6) -C (12) -175.93 (11)

C (9) -C (5) -C (6) -C (7) -174.98 (11)

C (4) -C (5) -C (6) -C (7) 5.40 (18)

C (5) -C (6) -C (7) -C (8) -39.51 (16)

C (12) -C (6) -C (7) -C (8) 141.86 (13)

C (1) -N (1) -C (8) -C (7) 152.41 (11)

C (4) -N (1) -C (8) -C (7) -32.29 (16)

C (6) -C (7) -C (8) -N (1) 51.19 (15)

C (6) -C (5) -C (9) -C (10) -1.21 (19)

C (4) -C (5) -C (9) -C (10) 178.41 (12)

C (13) -O (2) -C (10) -C (9) -26.55 (18)

C (13) -O (2) -C (10) -C (11) 155.32 (12)

C (5) -C (9) -C (10) -O (2) 178.92 (11)

C (5) -C (9) -C (10) -C (11) -3.02 (19)

C (14) -O (3) -C (11) -C (12) 7.02 (18)

C (14) -O (3) -C (11) -C (10) -173.21 (11)

O (2) -C (10) -C (11) -O (3) 3.21 (16)

C (9) -C (10) -C (11) -O (3) -175.02 (11)

O (2) -C (10) -C (11) -C (12) -177.01 (11)

C (9) -C (10) -C (11) -C (12) 4.76 (19)

O (3) -C (11) -C (12) -C (6) 177.47 (12)

C (10) -C (11) -C (12) -C (6) -2.28 (19)

C (5) -C (6) -C (12) -C (11) -1.94 (19)

07-21-2014

C (7) -C (6) -C (12) -C (11) 176.67 (12) C (2) -N (3) -C (15) -C (20) -72.70 (16 ) C (2) -N (3) -C (15) -C (16) 114.22 (14) C (20) -C (15) -C (16) -C (17) -0.3 ( 2) N (3) -C (15) -C (16) -C (17) 172.16 (12) C (20) -C (15) -C (16) -C (21) -178.86 (13) N (3) -C (15) -C (16) -C (21) -5.84 (19) C (15) -C (16) -C (17) -C (18) 0, 7 (2) C (21) -C (16) -C (17) -C (18) 179.27 (14) C (16) -C (17) -C (18) -C (19) -0 , 5 (2) C (16) -C (17) -C (18) -C (22) -179.15 (14) C (17) -C (18) -C (19) -C (20) -0.2 (2) C (22) -C (18) -C (19) -C (20) 178.52 (14) C (18) -C (19) -C (20) -C (15 ) 0.6 (2) C (18) -C (19) -C (20) -C (23) -175.26 (14) C (16) -C (15) -C (20) -C ( 19) -0.4 (2) N (3) -C (15) -C (20) -C (19) -173.37 (12) C (16) -C (15) -C (20) - C (23) 175.44 (13) N (3) -C (15) -C (20) -C (23) 2.4 (2) C (1) -N (2) -C (24) - C (25) 85.94 (13) C (2) -N (2) -C (24) -C (25) -89.49 (13) C (26) -N (4) -C (25) -C (24) -105.34 (4) N (2) -C (24) -C (25) -N (4) 177.45 (10) C (25) -N (4) -C (26 ) -O (4) -5.4 (2) C (25) -N (4) -C (26) -N (5) 173.13 (12)

Unless otherwise defined, all scientific and technical terms used herein are in accordance with the meaning known by the common expert in the field.

Claims (12)

E11748611 07-21-2014 1. A crystalline polymorph (I) of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1- to] isoquinolin-3 (4H) -yl] ethyl} urea) having an X-ray powder diffraction pattern comprising characteristic peaks, in terms of 2, at 10.1 ° and 12.9 °. 2. The polymorph of claim 1, wherein said powder X-ray diffraction pattern further comprises characteristic peaks, in terms of 2, at 15.3 ° and 17.6 °. The polymorph of claim 1, wherein said powder X-ray diffraction pattern comprises at least 5 characteristic peaks in terms of 2, selected from 6.4 °, 10.1 °, 12.6 °, 12 , 9th, 13.6th, 14.2th, 14.7th, 15.3th, 15.4th, 15.8th, 17.0th, 17.6th, 18.9th, 20.9th, 22.4th, 22.8th and 28.7º. 4. The crystalline polymorph (I) of any one of claims 1 to 3, having the following structural parameters obtained by means of individual crystal analysis: Wavelength 0.71073 angstrom Triclinic crystalline system Space group P-1 Unit cell dimensions a = 8.1246 (4) angstrom  = 91.583 (4) º. b = 11,4573 (5) angstrom  = 90,299 (4) º. c = 13.2398 (6) angstrom  = 99.628 (4) º. Volume 1214.56 (10) angstrom3 Z 2. 5. The polymorph of any one of claims 1 to 4, which has a trace calorimetry scan differential showing a maximum at 248 ° C. twenty 6. A solid composition comprising the polymorph of any one of claims 1 to 4, wherein at least 50% by weight of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy Total -4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present as said polymorph. The composition of claim 6, wherein at least 70% by weight of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro- Total 2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present as said polymorph. 8. The composition of claim 6, wherein at least 90% by weight of (N- {2 - [(2E) -2- (mesitylimino) -9.10 30 dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present as said polymorph. 9. The composition of claim 6, wherein at least 97% by weight of (N- {2 - [(2E) -2- (mesitylimino) -9.10 Dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea) of said composition is present as said polymorph. 10. A solid composition comprising the polymorph of any one of claims 1 to 4 and a pharmaceutically acceptable excipient. A solid composition comprising the polymorph of any one of claims 1 to 4, and a known therapeutic substance that is used to treat asthma, allergic asthma, pollen rhinitis, allergic rhinitis, bronchitis, chronic obstructive pulmonary disease (COPD). , acute adult dysneic syndrome (SDAA), cystic fibrosis, skin disorders, atopic dermatitis, psoriasis, eye inflammation, cerebral ischemia or autoimmune diseases. Four. Five

12.

 The solid composition of claim 11, wherein the therapeutic substance is used to treat asthma or COPD.

13.

A crystalline polymorph as defined in any one of claims 1 to 5, for use in the treatment of asthma, allergic asthma, pollinic rhinitis, allergic rhinitis, bronchitis, obstructive pulmonary disease

50 chronic (COPD), acute adult dyspnoea syndrome (SDAA), cystic fibrosis, skin disorders, atopic dermatitis, psoriasis, eye inflammation, cerebral ischemia or autoimmune diseases, in a mammal that needs it. 14. The crystalline polymorph for use in claim 13, wherein the mammal is a human. 55 29 E11748611 07-21-2014 15. A compound of (N- {2 - [(2E) -2- (mesitylimino) -9,10-dimethoxy-4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin3 (4H) -yl] ethyl} urea), in the form of a crystalline solid consisting of more than 99% by weight of (N- {2 - [(2E) -2 (mesitylimino) -9,10-dimethoxy) 4-oxo-6,7-dihydro-2H-pyrimido [6,1-a] -isoquinolin-3 (4H) -yl] ethyl} urea), at least 95% by weight in polymorphic form of the crystalline polymorph of a any of claims 1 to 4. 30